MicroRNAs靶向JAK-STAT信号通路调控RBSDV侵染的分子机制

MicroRNA (miRNA) were participate in the interaction between insect host and plant virus, but the investigations of their mechanism were still limited. To explore miRNAs functions on rice black-streaked dwarf virus (RBSDV) infection, our previous study used RBSDV and its insect host, Laodelphax striatellus, as research object to discover differentially expressed (DE) miRNAs in host midgut by high throughput sequencing. We found JAK-STAT signaling pathway was the most significantly affected one in KEGG pathway analysis of DE miRNAs. RBSDV infection significantly increase JAK-STAT related miRNAs (PC-5p-33 and PC-3p-70) expression, over expression of them in L. striatellus promoted RBSDV infection. To further investigate the mechanisms of JAK-STAT pathway related miRNAs in virus infection, we will conduct miRNA gain-of-function and loss-of-function assays by miRNA mimics and antagomirs injection, and clarified the regulatory effect of miRNAs on virus infection. RT-qPCR and Western blotting will be used to analyze the regulatory relationship between miRNAs and candidate target genes. Meanwhile, Dual luciferase reporter assay will be used to verify their physical interactions. RNA interference (RNAi) and immunofluorescence technology will be used to investigate the function of target genes on the suppression of JAK-STAT signaling pathway and RBSDV infection. At last, double interference of miRNAs and its functional target genes will be carried on to reveal the mechanism underlying the regulation of virus infection by JAK-STAT pathway which was mediated by miRNAs. The results are expected to explain the molecular mechanism of miRNA in regulating virus infection.

MicroRNA（miRNA）参与昆虫与植物病毒互作过程，但其作用机制研究较少。为解析miRNA在调控植物病毒侵染介体昆虫中的作用，我们以水稻黑条矮缩病毒（RBSDV）与灰飞虱互作体系为研究对象，对感染病毒的灰飞虱中肠进行miRNA测序，发现JAK-STAT通路为差异miRNAs调控的主要免疫途径。为解析miRNA调控JAK-STAT通路进而调控病毒侵染的机制，本研究拟通过显微注射模拟物和抑制剂，过表达和抑制JAK-STAT通路相关miRNA的表达，明确miRNA对病毒侵染的调控作用；通过分析靶基因与miRNA的表达调控关系，结合双荧光素酶报告系统，鉴定靶基因；利用RNAi和免疫荧光等技术明确靶基因对JAK-STAT通路及病毒侵染的影响；通过双重干扰miRNA和靶基因，明确miRNA是否通过靶基因调控JAK-STAT免疫途径，进而调控病毒的侵染。研究结果有望解析miRNA调控病毒侵染机制。

MicroRNA（miRNA）参与昆虫与植物病毒互作过程，但其作用机制研究较少。为解析miRNA在调控植物病毒侵染介体昆虫中的作用，我们以水稻黑条矮缩病毒（RBSDV）与灰飞虱互作体系为研究对象开展研究。由于介体昆虫的中肠是抵御植物病毒侵染的第一道屏障，因此通过对感染病毒的灰飞虱中肠进行miRNA高通量测序和实验验证，共发现19条差异miRNAs参与病毒侵染调控。本研究首次发现病毒侵染后上调的miR-184-3p和PC-5p-33_137169可促进病毒在介体昆虫体内的病毒积累量和中肠组织侵染。miR-184-3p和PC-5p-33_137169分别作用于靶标基因Ken和STAT5B，通过间接和直接作用于介体昆虫JAK-STAT信号通路发挥调控作用。通过分析miRNA与靶基因转录水平在过表达处理组和不同时间点的表达谱，结合双荧光素酶报告系统，我们首次鉴定Ken和STAT5B为miR-184-3p和PC-5p-33_137169的直接作用靶标。利用RNAi和免疫荧光等技术明确Ken和STAT5B敲减后会导致JAK-STAT通路被抑制。当JAK-STAT通路关键基因STAT5B的磷酸化被抑制，中断通路级联反应后，病毒复制和积累量上升。不同时间节点的定量PCR检测结果表明miR-184-3p和PC-5p-33_137169调控的时间窗口为病毒侵染中肠屏障的早期，即介体获毒后的2天至3天。本研究首次发现灰飞虱新的未被报道的miRNAs：PC-5p-33_137169和物种间保守的发育调控因子miR-184-3p在介体昆虫先天免疫中的新功能。本研究成果表明斐济病毒属病毒RBSDV在侵染介体昆虫中肠组织、突破中肠屏障的过程中，可以上调介体昆虫内源性miRNAs的表达，利用miRNAs的作用途径使其靶标基因对介体昆虫的先天免疫途径JAK-STAT信号通路发挥抑制作用，降低介体昆虫抗病毒的免疫能力，促进病毒在细胞中的复制，加快病毒在介体昆虫体内的侵染进程。该成果发现的新的miRNAs在介体昆虫与病毒的互作中的新功能，填补了灰飞虱miRNAs对植物病毒在其体内的复制和传播、先天免疫应答等分子调控机制上的功能研究空白。揭示了小分子RNA可受到病毒的影响进而作用于介体的抗病毒防御，对揭示植物、病毒与介体昆虫三者之间的互作致病机理具有重要意义。